Medicinal compositions for promoting recovery from stress loading and novel matsutake mushroom strain

ABSTRACT

A pharmaceutical composition for promoting a recovery from stress comprising  Tricholoma matsutake , a hot water extract of  Tricholoma matsutake  or a dried product thereof, or an alkaline solution extract of  Tricholoma matsutake  or a dried product thereof, and a pharmaceutically acceptable carrier; and a novel  Tricholoma matsutake  strain FERM BP-7304 are disclosed.

TECHNICAL FIELD

[0001] The present invention relates to a pharmaceutical composition forpromoting a recovery from stress and a novel Tricholoma matsutakestrain. The pharmaceutical composition of the present invention forpromoting a recovery from stress may be administered as a medicament orin various forms, for example, eatable or drinkable products such asfunctional foods or health foods, or feeds. Further, the pharmaceuticalcomposition of the present invention for promoting a recovery fromstress may be administered in the form of an oral hygienic composition,which is temporarily kept in the mouth but then spat out, retainingalmost none of the components, for example, a dentifrice, a mouthwashagent, a chewing gum, or a collutorium, or in the form of an inhalationdrawn in through the nose.

BACKGROUND ART

[0002] It is known that Tricholoma matsutake contains manyphysiologically active substances. For example, Japanese Examined PatentPublication (Kokoku) No. 57-1230 and Japanese Patent No. 2767521disclose various antitumor substances contained in Tricholoma matsutake.The above Japanese Examined Patent Publication (Kokoku) No. 57-1230discloses that emitanine-5-A, emitanine-5-B, emitanine-5-C, andemitanine-5-D, which are separated and purified from a liquid extractobtained by extracting a broth of Tricholoma matsutake mycelia with hotwater or a diluted alkaline solution, exhibits an activity of inhibitinga proliferation of sarcoma 180 cells. The above Japanese Patent No.2767521 discloses that a protein with a molecular weight of 0.2 to 0.21million (a molecular weight of a subunit=0.1 to 0.11 million) which isseparated and purified from an extract of Tricholoma matsutake fruitbodies with water exhibits an antitumor activity.

[0003] Further, the present inventor found that a hot water extract ofTricholoma matsutake or an alkaline solution extract of Tricholomamatsutake, or an adsorption fraction of a hot water extract ofTricholoma matsutake or an alkaline solution extract of Tricholomamatsutake by an anion exchange resin exhibits an immuno-enhancingactivity (Japanese Patent Application No. 2000-374).

[0004] It is known that stress not only causes a disorder in anadjustment of social life, such as psychosomatic disorder, but also maybe a trigger of an attack and a progress of life-style related diseasesor the like. An antianxiety agent (tranquilizer) or herbal medicines areprescribed for changes of mind and body caused by stress, whereas amethod for effectively treating changes of an immune function caused bystress has not yet been found. To the present inventor's knowledge, nofood having an activity of promoting a spontaneous recovery of an immuneactivity by a release of stress (i.e., activity of promoting a recoveryfrom stress) is known. Further, to the present inventor's knowledge, itis not known that a substance having the activity of promoting arecovery from stress exists in food.

[0005] The present inventor made an intensive search for anotherphysiological activity other than the known antitumor activity orimmuno-enhancing activity in Tricholoma matsutake. As a result, thepresent inventor newly found that an active ingredient exhibiting theactivity of promoting a recovery from stress is contained in Tricholomamatsutake. Further, the present inventor made an intensive search for aphysiological activity of a novel Tricholoma matsutake strain found bythe present inventor, and found a novel Tricholoma matsutake straincontaining an active ingredient exhibiting an excellent activity ofpromoting a recovery from stress. The present invention is based on theabove findings.

DISCLOSURE OF INVENTION

[0006] This present invention relates to a pharmaceutical compositionfor promoting a recovery from stress, comprising Tricholoma matsutake[Tricholoma matsutake (S. Ito & Imai) Sing.], a hot water extract ofTricholoma matsutake or a dried product thereof, or an alkaline solutionextract of Tricholoma matsutake or a dried product thereof, and apharmaceutically acceptable carrier.

[0007] Further, the present invention relates to a functional food forpromoting a recovery from stress, comprising Tricholoma matsutake, a hotwater extract of Tricholoma matsutake or a dried product thereof, or analkaline solution extract of Tricholoma matsutake or a dried productthereof, alone or optionally with one or more food components.

[0008] Further, the present invention relates to an oral hygieniccomposition for promoting a recovery from stress, comprising Tricholomamatsutake, a hot water extract of Tricholoma matsutake or a driedproduct thereof, or an alkaline solution extract of Tricholoma matsutakeor a dried product thereof, and a carrier for an oral hygieniccomposition.

[0009] Further, the present invention relates to a method for promotinga recovery from stress, comprising administering to a subject in needthereof Tricholoma matsutake, a hot water extract of Tricholomamatsutake or a dried product thereof, or an alkaline solution extract ofTricholoma matsutake or a dried product thereof, in an amount effectivetherefor.

[0010] Further, the present invention relates to the use of Tricholomamatsutake, a hot water extract of Tricholoma matsutake or a driedproduct thereof, or an alkaline solution extract of Tricholoma matsutakeor a dried product thereof, in the manufacture of a pharmaceuticalcomposition for promoting a recovery from stress, a functional food forpromoting a recovery from stress, or an oral hygienic composition forpromoting a recovery from stress.

[0011] Further, the present invention relates to a Tricholoma matsutakestrain FERM BP-7304, or a mycelium, a broth, or a fruit body of aTricholoma matsutake strain FERM BP-7304.

[0012] Further, the present invention relates to a hot water extract ofa Tricholoma matsutake strain FERM BP-7304 or a dried product thereof,or an alkaline solution extract of a Tricholoma matsutake strain FERMBP-7304 or a dried product thereof.

BRIEF DESCRIPTION OF DRAWINGS

[0013]FIG. 1 is a photograph showing the results of electrophoresis ofPCR products obtained by a RAPD method using known Tricholoma matsutakestrains represented by Nos. 1 to 6 in Table 1.

[0014]FIG. 2 is a photograph showing the results of electrophoresis ofPCR products obtained by a RAPD method using known Tricholoma matsutakestrains represented by Nos. 7 to 11 in Table 1.

[0015]FIG. 3 is a photograph showing the results of electrophoresis ofPCR products obtained by a RAPD method using the Tricholoma matsutakestrain FERM BP-7304 of the present invention and known Tricholomamatsutake strains represented by Nos. 12 to 13 in Table 1.

[0016]FIG. 4 is a photograph showing the results of electrophoresis ofPCR products obtained by a RAPD method using the Tricholoma matsutakestrain FERM BP-7304 of the present invention.

[0017]FIG. 5 is a graph showing an activity of promoting a recovery fromstress in the mixture of the dry powder of a hot water extract ofTricholoma matsutake strain FERM BP-7304 of the present invention andthe dry powder of an alkaline solution extract thereof at a ratio of3.2:5.1.

[0018]FIG. 6 is a graph showing an activity of promoting a recovery fromstress in each dry powder of the Tricholoma matsutake strain FERMBP-7304 of the present invention and Tricholoma matsutake strains.

[0019]FIG. 7 is a graph showing an activity of promoting a recovery fromstress in each dry powder of other Tricholoma matsutake strains.

[0020]FIG. 8 is a graph showing an activity of promoting a recovery fromstress in each dry powder of still other Tricholoma matsutake strains.

[0021]FIG. 9 is a graph showing an activity of promoting a recovery fromstress in each dry powder of still other Tricholoma matsutake strains.

BEST MODE FOR CARRYING OUT THE INVENTION

[0022] The present invention will be explained in detail hereinafter.

[0023] The pharmaceutical composition of the present invention forpromoting a recovery from stress contains as an active ingredient atleast one of

[0024] (1) Tricholoma matsutake (for example, a mycelium, a broth, or afruit body of Tricholoma matsutake);

[0025] (2) a hot water extract of Tricholoma matsutake (for example, ahot water extract of the mycelium, the broth, or the fruit body ofTricholoma matsutake) or a dried product thereof; or

[0026] (3) an alkaline solution extract of Tricholoma matsutake (forexample, an alkaline solution extract of the mycelium, the broth, or thefruit body of Tricholoma matsutake) or a dried product thereof;

[0027] and a pharmaceutically or veterinarily acceptable ordinarycarrier or diluent.

[0028] As the Tricholoma matsutake used for preparing the Tricholomamatsutake, the hot water extract of Tricholoma matsutake or the driedproduct thereof, or the alkaline solution extract of Tricholomamatsutake or the dried product thereof as the active ingredient of thepresent invention, there may be mentioned, for example, a fruit body ora mycelium of a naturally occurring Tricholoma matsutake, or a mycelium(i.e., a cultured mycelium), a broth, or a fruit body obtainable byculturing Tricholoma matsutake. The novel Tricholoma matsutake strainFERM BP-7304 [Tricholoma matsutake (S. Ito & Imai) Sing. CM6271] of thepresent invention is preferably used, because the strain contains theactive ingredient exhibiting an excellent activity of promoting arecovery from stress.

[0029] As the mycelium of Tricholoma matsutake which may be used as theactive ingredient in the pharmaceutical composition of the presentinvention for promoting a recovery from stress, when the myceliaobtainable by cultivation are used, for example, wet mycelia obtained byremoving medium from a mixture of the mycelia obtainable by cultivation(i.e., the cultured mycelia) and the medium by an appropriate removingmethod (such as filtration), dried mycelia obtained by further removingwater from the wet mycelia by an appropriate method (such aslyophilization), or powdery dried mycelia obtained by further crushingthe dried mycelia may be used. When the naturally occurring mycelia areused, for example, the naturally occurring mycelia without anytreatment, dried mycelia obtained by removing water from the naturallyoccurring mycelia by an appropriate method (such as lyophilization), orpowdery dried mycelia obtained by further crushing the dried mycelia maybe used.

[0030] As the broth of Tricholoma matsutake which may be used as theactive ingredient in the pharmaceutical composition of the presentinvention for promoting a recovery from stress, for example, a mixtureof the mycelia obtainable by cultivation (i.e., the cultured mycelia)and a medium, a dried broth obtained by removing water from the mixtureby an appropriate method (such as lyophilization), or a powdery driedbroth obtained by further crushing the dried broth may be used.

[0031] As the fruit body of Tricholoma matsutake which may be used asthe active ingredient in the pharmaceutical composition of the presentinvention for promoting a recovery from stress, for example, fruitbodies obtainable by cultivation or naturally occurring fruit bodieswithout any treatment, crushed fruit bodies obtained by crushing thefruit bodies, dried fruit bodies obtained by removing water from thefruit bodies by an appropriate method (such as lyophilization), orpowdery dried fruit bodies obtained by further crushing the dried bodiesmay be used.

[0032] The hot water extract of Tricholoma matsutake may be prepared,for example, by extracting fruit bodies or mycelia of a naturallyoccurring Tricholoma matsutake, or mycelia (i.e., cultured mycelia), abroth, or fruit bodies obtainable by culturing Tricholoma matsutake withhot water.

[0033] A temperature of hot water used in the hot water extracting stepis not limited, so long as the active ingredient exhibiting an activityof promoting a recovery from stress is sufficiently extracted fromTricholoma matsutake in the hot water extract, but is preferably 60 to100° C., more preferably 80 to 98° C.

[0034] When mycelia or fruit bodies are used in the hot water extractingstep, it is preferable to crush, grind, or pulverize them, to enhancethe extraction efficiency.

[0035] It is preferable to carry out the hot water extracting step withstirring or shaking, so that an extraction efficiency is enhanced. Anextracting time may vary with the form of Tricholoma matsutake, forexample, a phase of fruit bodies, mycelia, or a broth, a treated form,such as a crushed, ground, or pulverized form, a temperature of hotwater, or a treating condition with or without stirring or shrinking,but is for example, 1 to 6 hours, preferably 2 to 3 hours.

[0036] The resulting hot water extract liquor may be used as it is,namely, in the state containing insolubles, as the active ingredient ofthe pharmaceutical composition of the present invention for promoting arecovery from stress. Alternatively, it may be used after removing theinsolubles, or after removing the insolubles and then low molecularweight fractions from the extract liquor, as the active ingredient ofthe pharmaceutical composition of the present invention for promoting arecovery from stress. For example, the insolubles may be removed bycentrifuging the hot water extract containing such insolubles, and theresulting supernatant may be used as the active ingredient of thepharmaceutical composition of the present invention for promoting arecovery from stress. Alternatively, after removing the insolubles bycentrifuging the hot water extract containing such insolubles, anddialyzing the resulting supernatant to remove low molecular weightfractions, preferably fractions of low molecular weight substanceshaving a molecular weight of 3500 or less, the resulting liquor may beused as the active ingredient of the pharmaceutical composition of thepresent invention for promoting a recovery from stress. Further, afterremoving water from the hot water extract liquor (for example, the hotwater extract liquor containing insolubles, the supernatant obtainableby centrifuging the hot water extract liquor, or the liquor obtainableby dialyzing the supernatant) by an appropriate method (such aslyophilization), the resulting dried product may be used as the activeingredient of the pharmaceutical composition of the present inventionfor promoting a recovery from stress.

[0037] The alkaline solution extract of Tricholoma matsutake or thedried product thereof, as the active ingredient of the pharmaceuticalcomposition of the present invention for promoting a recovery fromstress, may be prepared by, for example, a method similar to that forpreparing the hot water extract of Tricholoma matsutake as mentionedabove. More particularly, the procedures of the above-mentioned methodfor preparing the hot water extract of Tricholoma matsutake can berepeated except that an alkaline solution is used instead of hot water,to prepare the alkaline solution extract of Tricholoma matsutake. Forexample, it may be obtained by extracting fruit bodies or mycelia of anaturally occurring Tricholoma matsutake, or mycelia (i.e., culturedmycelia), a broth, or fruit bodies obtainable by culturing Tricholomamatsutake with an alkaline solution.

[0038] The alkaline solution used in the alkaline solution-extractingstep may be, for example, but is by no means limited to, an aqueoussolution of a hydroxide of an alkaline metal, such as sodium orpotassium, particularly sodium hydroxide. A pH value of the alkalinesolution is preferably 8 to 13, more preferably 9 to 12. The alkalinesolution-extracting step is carried out preferably at 0 to 30° C., morepreferably at 0 to 25° C. The resulting alkaline solution extract may beused as the active ingredient of the pharmaceutical composition of thepresent invention for promoting a recovery from stress, without anytreatment such as neutralization, or after neutralization.

[0039] The pharmaceutical composition of the present invention forpromoting a recovery from stress can be administered to an animal,preferably a mammal, more preferably a human, in the form of a mixtureof Tricholoma matsutake (for example, a mycelium, a broth, or a fruitbody of Tricholoma matsutake), the hot water extract of Tricholomamatsutake (for example, a hot water extract of a mycelium, a broth, or afruit body of Tricholoma matsutake) or the dried product thereof, or thealkaline solution extract of Tricholoma matsutake (for example, analkaline solution extract of a mycelium, a broth, or a fruit body ofTricholoma matsutake) or the dried product thereof, with apharmaceutically or veterinarily acceptable ordinary carrier or diluent.

[0040] The active ingredient of the pharmaceutical composition of thepresent invention for promoting a recovery from stress, that is,Tricholoma matsutake, the hot water extract of Tricholoma matsutake orthe dried product thereof, or the alkaline solution extract ofTricholoma matsutake or the dried product thereof, exhibits an activityof promoting a recovery from stress.

[0041] The active ingredient of the present invention, that is,Tricholoma matsutake, the hot water extract of Tricholoma matsutake orthe dried product thereof, or the alkaline solution extract ofTricholoma matsutake or the dried product thereof, can be administeredto a subject in need of promoting a recovery from stress, with orwithout, but preferably with, a pharmaceutically or veterinarilyacceptable ordinary carrier or diluent, in an amount effective therefor.

[0042] Further, the active ingredient of the present invention, that is,Tricholoma matsutake, the hot water extract of Tricholoma matsutake orthe dried product thereof, or the alkaline solution extract ofTricholoma matsutake or the dried product thereof, can be used in themanufacture of a pharmaceutical composition for promoting a recoveryfrom stress, a functional food for promoting a recovery from stress, oran oral hygienic composition for promoting a recovery from stress.

[0043] Generally, when stress is loaded to an animal once or during acertain period, an immune activity in the animal is decreased, but theimmune activity is spontaneously recovered after a release of thestress. The “activity of promoting a recovery from stress” as usedherein means an activity which promotes the recovery of an immuneactivity during a period of an immune activity convalescence after arelease of stress, in comparison with the spontaneous recovery.

[0044] The pharmaceutical composition of the present invention forpromoting a recovery from stress can be administered at any time, solong as it can promote the recovery of an immune activity brieflylowered by stress. It can be administered, for example, before a loadingof stress, during a loading of stress, and/or during a period of animmune activity convalescence after a release of stress.

[0045] In this connection, the “activity of promoting a recovery fromstress” in the present invention is different from the above-describedmere “immuno-enhancing activity” previously found by the presentinventor. Generally, the “immuno-enhancing activity” means an activityin which an enhancement of an immune activity is observed byadministering an active ingredient having such an activity in comparisonwith a state before the administration, that is, an activity ofenhancing an immune activity per se. The state before the administrationmay be a state in which an immune activity is natural or lowered bystress. In contrast, the “activity of promoting a recovery from stress”in the present invention is an activity which promotes the recovery ofan immune activity during a period of an immune activity convalescence,as described above, that is, an activity of enhancing the speed ofrecovery of an immune activity. When the pharmaceutical composition ofthe present invention for promoting a recovery from stress isadministered, the speed of recovery of an immune activity is increasedin comparison with the case in which the pharmaceutical composition ofthe present invention for promoting a recovery from stress is notadministered.

[0046] Further, in the “immuno-enhancing activity”, enhancement of animmune activity is directly observed when administering an activeingredient having such an activity. In contrast, when administering theactive ingredient of the pharmaceutical composition of the presentinvention for promoting a recovery from stress (i.e., Tricholomamatsutake, the hot water extract of Tricholoma matsutake or the driedproduct thereof, or the alkaline solution extract of Tricholomamatsutake or the dried product thereof) to a subject animal before aloading of stress, the recovery of an immune activity is promoted duringa period of an immune activity convalescence, even if the activeingredient is not administered during a loading of stress and during aperiod of an immune activity convalescence. With respect to the point,the “activity of promoting a recovery from stress” in the presentinvention is different from the “immuno-enhancing activity”.

[0047] The formulation of the pharmaceutical composition of the presentinvention for promoting a recovery from stress is not particularlylimited to, but may be, for example, oral medicines, such as powders,fine particles, granules, tablets, capsules, suspensions, emulsions,syrups, extracts or pills, or parenteral medicines, such as injections,liquids for external use, ointments, suppositories, creams for topicalapplication, or eye lotions.

[0048] The oral medicines may be prepared by an ordinary method using,for example, fillers, binders, disintegrating agents, surfactants,lubricants, flowability-enhancers, diluting agents, preservatives,coloring agents, perfumes, tasting agents, stabilizers, humectants,antiseptics, antioxidants or the like, such as gelatin, sodium alginate,starch, corn starch, saccharose, lactose, glucose, mannitol,carboxylmethylcellulose, dextrin, polyvinyl pyrrolidone, crystallinecellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesiumstearate, polyethylene glycol, magnesium silicate, silicic anhydride, orsynthetic aluminum silicate.

[0049] The parenteral administration may be, for example, an injectionsuch as a subcutaneous or intravenous injection, or a per rectumadministration. Of the parenteral formulations, an injection ispreferably used.

[0050] When the injections are prepared, for example, water-solublesolvents, such as physiological saline or Ringer's solution,water-insoluble solvents, such as plant oil or fatty acid ester, agentsfor rendering isotonic, such as glucose or sodium chloride, solubilizingagents, stabilizing agents, antiseptics, suspending agents, oremulsifying agents may be optionally used, in addition to the activeingredient.

[0051] The pharmaceutical composition of the present invention forpromoting a recovery from stress may be administered in the form of asustained release preparation using sustained release polymers. Forexample, the pharmaceutical composition of the present invention forpromoting a recovery from stress may be incorporated to a pellet made ofethylenevinyl acetate polymers, and the pellet may be surgicallyimplanted in a tissue to be treated.

[0052] The pharmaceutical composition of the present invention forpromoting a recovery from stress may contain Tricholoma matsutake, thehot water extract of Tricholoma matsutake or the dried product thereof,or the alkaline solution extract of Tricholoma matsutake or the driedproduct thereof in an amount of, but is by no means limited to, 0.01 to99% by weight, preferably 0.1 to 80% by weight.

[0053] A dose of the pharmaceutical composition of the present inventionfor promoting a recovery from stress is not particularly limited, butmay be determined dependent upon the kind of disease, the age, sex, bodyweight, or symptoms of the subject, a method of administration, or thelike. The immuno-enhancing composition of the present invention may beorally or parenterally administered.

[0054] The pharmaceutical composition of the present invention forpromoting a recovery from stress may be administered as a medicament orin various forms, for example, eatable or drinkable products, such asfunctional foods or health foods, or feeds. Further, the pharmaceuticalcomposition of the present invention for promoting a recovery fromstress may be administered in the form of an oral hygienic composition,which is temporarily kept in the mouth, but then spat out, retainingalmost none of the components, for example, a dentifrice, a mouthwashagent, a chewing gum, or a collutorium, or in the form of an inhalationdrawn in through the nose. For example, Tricholoma matsutake, the hotwater extract of Tricholoma matsutake or the dried product thereof, orthe alkaline solution extract of Tricholoma matsutake or the driedproduct thereof may be added to a desired food including a drink, afeed, a dentifrice, a mouthwash agent, a chewing gum, a collutorium, orthe like as an additive, such as a food additive.

[0055] The Tricholoma matsutake strain FERM BP-7304 was deposited in theInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology [(Former Name) National Institute ofBioscience and Human-Technology Agency of Industrial Science andTechnology (Address: AIST Tsukuba Central 6, 1-1, Higashi 1-chomeTukuba-shi, Ibaraki-ken 305-8566 Japan)] on Sep. 14, 2000. TheTricholoma matsutake strain FERM BP-7304 was established as a strain byculturing a piece of fruit body from a Tricholoma matsutake strainCM6271 collected in Kameoka-shi, Kyoto, Japan and then culturing it invitro, and is maintained in the Biomedical Research Laboratories, KurehaChemical Industry Co. Ltd.

[0056] The macroscopical observation of a fruit body of the Tricholomamatsutake strain FERM BP-7304 of the present invention is identical withthat of a Tricholoma matsutake fruit body described in Rokuya Imasekiand Tsuguo Hongo, “Gensyoku Nihon Shinkinrui Zukan (ColoredIllustrations of Mushrooms of Japan) (I)”, Hoiku-sha (Osaka, Japan),1987, plate 15 and page 77. The Tricholoma matsutake strain FERM BP-7304of the present invention can be subcultivated or maintained on aMATSUTAKE medium. A large-scale cultivation of mycelia of the Tricholomamatsutake strain FERM BP-7304 can be carried out by inoculating thestrain into a liquid medium and then performing, for example, a staticculture, a shake culture, or a tank culture.

[0057] When mycelia of the Tricholoma matsutake strain FERM BP-7304 ofthe present invention are inoculated on a MATSUTAKE medium, white hyphaegrow radially and thickly, and form large colonies. According to anobservation by a scanning electron microscope, a large number ofbranched mycelia having a diameter of 1 to 2 μm exist and projectionshaving a height of approximately 2 to 3 μm are observed at the side ofmycelia. In this connection, the Tricholoma matsutake strain FERMBP-7304 can be subcultured or cultured, generally in the form ofmycelia, but sometimes in the form of fruit bodies.

[0058] Mycological features of the Tricholoma matsutake strain FERMBP-7304 of the present invention will be explained hereinafter.

[0059] (1) Cultural and Morphological Features on Malt Extract AgarMedium

[0060] When the Tricholoma matsutake strain FERM BP-7304 of the presentinvention is inoculated on a malt extract agar medium, white hyphae growradially and thickly, and form colonies. A diameter of the colony after30 days from the inoculation is approximately 4 cm.

[0061] (2) Cultural and Morphological Features on Potato and GlucoseAgar Medium, Czapek's Agar Medium, Sabouraud Agar Medium, Oatmeal AgarMedium, Synthetic Mucor Agar Medium, and Medium for Assaying PhenolOxidase Reaction

[0062] When the Tricholoma matsutake strain FERM BP-7304 of the presentinvention is inoculated on a potato and glucose agar medium, a Czapek'sagar medium, a sabouraud agar medium, an oatmeal agar medium, asynthetic mucor agar medium, or a medium for assaying a phenol oxidasereaction, a growth of hyphae is not observed after a month from theinoculation.

[0063] (3) Cultural and Morphological Features on YpSs Agar Medium

[0064] The Tricholoma matsutake strain FERM BP-7304 of the presentinvention grows as a mat-like mycelia having a white gloss on a YpSsagar medium. A growth area after 30 days from the inoculation isapproximately 5 mm in radius.

[0065] (4) Cultural and Morphological Features on Glucose and Dry YeastAgar Medium

[0066] The Tricholoma matsutake strain FERM BP-7304 of the presentinvention grows as a mat-like mycelia having white gloss on a glucoseand dry yeast agar medium. A growth distance after 30 days from theinoculation is approximately 2 mm.

[0067] (5) Optimal Growth Temperature and Range of Temperature

[0068] After 100 mL-conical flasks each containing 10 mL of sterilemedium (3% glucose and 0.3% yeast extract, pH 7.0) were inoculated withapproximately 2 mg of a piece of the Tricholoma matsutake strain FERMBP-7304 mycelia of the present invention, cultivation was performed atvarious temperatures from 5 to 35° C. After the cultivation for 28 days,mycelia were taken from the flasks, washed thoroughly with distilledwater, and dried, and then each weight of mycelia was measured. As aresult, the weight of mycelia increased linearly at a range of 5 to 15°C., and gently at a range of 15 to 25° C. Mycelia did not grow at 27.5°C. or more. The optimal growth temperature is 15 to 25° C.

[0069] (6) Optimal Growth pH and Range of pH

[0070] A growth pH value was examined by preparing various media havinga pH in a range of 3.0 to 8.0. These media were prepared by adjusting apH of a liquid medium (3% glucose and 0.3% yeast extract) with 1 mol/LHCL or 1 mol/L potassium hydroxide. Each medium was sterilized through afilter, and then 10 mL of the sterile medium was poured into a 100mL-conical flask (previously sterilized). After approximately 2 mg of apiece of the Tricholoma matsutake strain FERM BP-7304 mycelia of thepresent invention was inoculated, cultivation was performed at 22° C.After the cultivation for 28 days, mycelia were taken from the flasks,washed thoroughly with distilled water, and dried, and then each weightof mycelia was measured. As a result, the limit for growth of myceliawas in a range of a pH 3.0 to 7.0. the optimal growth pH was 4.0 to 6.0.

[0071] (7) Formation of Confront Line by Confrontation Culture

[0072] A block (approximately 3 mm×3 mm×3 mm) of the Tricholomamatsutake strain FERM BP-7304 of the present invention and each block(approximately 3 mm×3 mm×3 mm) of 13 kinds of Tricholoma matsutakestrains listed in Table 1 described below were inoculated with a spaceof approximately 2 cm therebetween on a MATSUTAKE medium. After acultivation was carried out at 22° C. for 3 weeks, it was observedwhether or not a zone appeared at the boundary between two colonies.

[0073] As a result, the Tricholoma matsutake strain FERM BP-7304 of thepresent invention did not form a clear zone with respect to the 13 kindsof Tricholoma matsutake strains listed in Table 1. In this connection,it is considered that Tricholoma matsutake does not form a zone in aconfrontation culture. Among the 13 kinds of Tricholoma matsutakestrains listed in Table 1, no combinations formed a clear zone.

[0074] (8) Auxotrophy

[0075] After a 100 mL-conical flask containing 10 mL of a sterilesynthetic medium for mycorrhizae (Ohta et al., Trans. Mycol. Soc. Jpn.,31, 323, 1990) was inoculated with approximately 2 mg of a piece of theTricholoma matsutake strain FERM BP-7304 mycelia of the presentinvention, cultivation was performed at 22° C. After the cultivation for42 days, mycelia were taken from the flask, washed thoroughly withdistilled water, and dried, and then the weight of mycelia was measuredto obtain 441 mg of mycelia.

[0076] Each medium in which one of 28 kinds of sugar-related substanceswas substituted for glucose as a carbon (C) source in the syntheticmedium for mycorrhizae was inoculated with the Tricholoma matsutakestrain FERM BP-7304 of the present invention, and cultivation wasperformed. After the cultivation, each weight of mycelia was measured.

[0077] As a result, an order of the sugar-related substances from thatwhich achieved the heaviest weight to that which achieved the lightestweight was as follows:

[0078] wheat starch>cornstarch>dextrin>methyl-β-glucoside>cellobiose>mannose>fructose>arabinose>sorbitol>glucose>lactose>glycogen>mannitol>ribose>maltose>trehalose>galactose>raffinose>melibiose>N-acetylglucosamine.

[0079] In this connection, mycelia did not grow in each mediumcontaining cellulose, dulcitol, sucrose, xylose, methyl-α- glucoside,inulin, inositol, or sorbose.

[0080] Further, each medium in which one of 15 kinds of nitrogen-relatedsubstances was substituted for ammonium tartrate as a nitrogen (N)source in the synthetic medium for mycorrhizae was inoculated with theTricholoma matsutake strain FERM BP-7304 of the present invention, andcultivation was performed. After the cultivation, each weight of myceliawas measured.

[0081] As a result, an order of the nitrogen-related substances fromthat which achieved the heaviest weight to that which achieved thelightest weight was as follows:

[0082] corn steep liquor>soybean peptone>milk peptone>ammoniumnitrate>ammonium sulfate>ammonium tartrate>ammoniumcarbonate>asparagine>ammonium phosphate>ammonium chloride>sodiumnitrate>meat extract>yeast extract>casaminoacids>chlorella>tryptone>potasium nitrate.

[0083] Furthermore, each medium in which one component among mineralsand vitamins in the above synthetic medium was removed was inoculatedwith the Tricholoma matsutake strain FERM BP-7304 of the presentinvention, and cultivation was performed. After the cultivation, eachweight of mycelia was measured.

[0084] As a result, a deficiency of any one of calcium chloridedihydrate, manganese sulfate (II) pentahydrate, zinc sulfateheptahydrate, cobalt sulfate heptahydrate, copper sulphate pentahydrate,nickel sulfate hexahydrate, thiamin hydrochloride, nicotinic acid, folicacid, biotin, pyridoxine hydrochloride, carnitine chloride, adeninesulfate dihydrate, or choline hydrochloride did not affect the weight ofmycelia. In contrast, when any one of magnesium sulfate heptahydrate,iron chloride (II), or sodium dihydrogen phosphate was removed, theweight of mycelia was remarkably lowered. From the results, it isconsidered that magnesium, iron, phosphorus, and potassium are essentialfor the growth of the Tricholoma matsutake strain FERM BP-7304 of thepresent invention.

[0085] (9) Base Composition of DNA (GC Content)

[0086] The GC content of the Tricholoma matsutake strain FERM BP-7304 ofthe present invention is 49.9% (see Analytic Example 2 described below).

[0087] (10) DNA Patterns Generated by a RAPD Method

[0088] With respect to each DNA pattern generated by a RAPD (randomamplified polymorphic DNA) method using any one of six kinds of primers(10 mer; the actual base sequences are shown in Analytical Example 1described below) for PCR (polymerase chain reaction), the Tricholomamatsutake strain FERM BP-7304 of the present invention was compared with44 kinds of Tricholoma matsutake strains (the actual strains are shownin Analytical Example 1 described below). As a result, it was confirmedthat DNA patterns of the Tricholoma matsutake strain FERM BP-7304 of thepresent invention differed from those of 44 kinds of Tricholomamatsutake strains.

EXAMPLES

[0089] The present invention now will be further illustrated by, but isby no means limited to, the following Examples.

Example 1

[0090] Preparation of Dry Powder of Tricholoma matsutake Strain FERMBP-7304

[0091] After 500 mL-conical flasks (10 flasks) each containing 100 mL ofsterile medium (3% glucose and 0.3% yeast extract, pH 6.0) wereinoculated with mycelia of Tricholoma matsutake strain FERM BP-7304,cultivation was performed in a shaking incubator (250 rpm) at 22° C. for4 weeks. The resulting broth was filtered with a filter cloth toseparate mycelia. The mycelia were washed with distilled water. Afterhaving been frozen at −60° C., the mycelia were lyophilized using alyophilizer (MINIFAST MOD. DO. 5; Edwards) to obtain 10.1 g of driedmycelia. The resulting dried mycelia were crushed by a homoblender(Wonder Blender; Osaka Chemical Co., Ltd.) to obtain 9.8 g of drypowder.

Example 2

[0092] Preparation of Dry Powder of Hot Water Extract and AlkalineSolution Extract of Tricholoma matsutake Strain FERM BP-7304

[0093] After 15 g of dry powder of Tricholoma matsutake strain FERMBP-7304 mycelia prepared in a similar manner as that described inExample 1 and 600 mL of purified water were charged in a 1 L-beaker, anextraction treatment was performed in a water bath at 93 to 98° C. for 3hours while stirring. After the extraction was completed, the whole wascooled to room temperature and centrifuged at 12,000 rpm for 20 minutesto obtain a supernatant.

[0094] To the remaining pellets, 300 mL of purified water was added, andthe same procedures as above were performed. These procedures wererepeated three times. Supernatants obtained by each procedure and thesupernatant previously obtained were combined. The resulting mixture wasput into a dialysis membrane with a fractioning molecular weight of 3500(Spetra/Por 3 Membrane), and dialyzed in flowing tap water for 2 days.The inner part of the dialyzate was concentrated by a rotary evaporatorand lyophilized to obtain 3.2 g of dry powder of a hot water extract.

[0095] To the pellets remaining after the hot water extractiontreatment, 400 mL of 0.5 mol/L sodium hydroxide solution was added.Extraction was performed at 25° C. for 1 hour while stirring. Afterextraction, the whole was centrifuged (12,000 rpm, 20 minutes) to obtaina supernatant.

[0096] To the remaining pellets, 400 mL of 1.0 mol/L sodium hydroxidesolution was added. The same procedures as above were performed toobtain a supernatant. Two supernatants were combined, and the pH of thesupernatant was adjusted to 7.0 by adding 1.0 mol/L HCl thereto. Thewhole was put into a dialysis membrane (Spetra/Por 3 Membrane,fractioning molecular weight=3500), and dialyzed in flowing tap waterfor 2 days. The inner part of the dialyzate was concentrated by a rotaryevaporator and lyophilized to obtain 5.1 g of dry powder of an alkalinesolution extract.

Example 3

[0097] Preparation of Dry Powder of Tricholoma matsutake Fruit Bodiesfrom Iwate Prefecture

[0098] After 250 g of fruit bodies of commercially available Tricholomamatsutake harvested in Iwate Prefecture (purchased at Kuroshio market;Kita-Shinjuku, Shinjuku-ku, Tokyo) were lyophilized using a lyophilizer(MINIFAST MOD. DO. 5; Edwards) to remove water, the lyophilized fruitbodies were crushed by a homoblender (Wonder Blender; Osaka ChemicalCo., Ltd.) to obtain 35 g of dry powder.

Example 4

[0099] Preparation of Dry Powder of Hot Water Extract and AlkalineSolution Extract of Tricholoma matsutake from Iwate Prefecture

[0100] After 20 g of dry powder of commercially available Tricholomamatsutake fruit bodies from Iwate Prefecture prepared in a similarmanner as that described in Example 3 and 800 mL of purified water werecharged in 1 L-beaker, an extraction treatment was performed in a waterbath at 93 to 98° C. for 3 hours while stirring. After the extractionwas completed, the whole was cooled to room temperature and centrifugedat 12,000 rpm for 20 minutes to obtain a supernatant.

[0101] To the remaining pellets, 500 mL of purified water was added, andthe same procedures as above were performed. These procedures wererepeated three times. Supernatants obtained by each procedure and thesupernatant previously obtained were combined. The resulting mixture wasput into a dialysis membrane with a fractioning molecular weight of 3500(Spetra/Por 3 Membrane), and dialyzed in flowing tap water for 2 days.The inner part of the dialyzate was concentrated by a rotary evaporatorand lyophilized to obtain 1.0 g of dry powder.

[0102] To the pellets remaining after the hot water extractiontreatment, 500 mL of 0.5 mol/L sodium hydroxide solution was added.Extraction was performed at 25° C. for 1 hour while stirring. Afterextraction, the whole was centrifuged (12,000 rpm, 20 minutes) to obtaina supernatant.

[0103] To the remaining pellets, 500 mL of 1.0 mol/L sodium hydroxidesolution was added. The same procedures as above were performed toobtain a supernatant. Two supernatants were combined, and the pH of thesupernatant was adjusted to 7.0 by adding 1.0 mol/L HCl thereto. Thewhole was put into a dialysis membrane (Spetra/Por 3 Membrane,fractioning molecular weight=3500), and dialyzed in flowing tap waterfor 2 days. The inner part of the dialyzate was concentrated by a rotaryevaporator and lyophilized to obtain 5.1 g of dry powder of an alkalinesolution extract.

Example 5

[0104] Preparation of Dry Powder of Hot Water Extract of Tricholomamatsutake Fruit Bodies from Nagano Prefecture

[0105] The procedures described in Example 4 were repeated, except thatTricholoma matsutake fruit bodies harvested in Nagano Prefecture wereused instead of those harvested in Iwate Prefecture, to obtain 1.5 g ofdry powder of a hot water extract of Tricholoma matsutake fruit bodies.

Comparative Example 1

[0106] Preparation of Dry Powder of Hot Water Extract of Agaricus blazeiFruit Bodies

[0107] The procedures described in Example 4 were repeated, except thatcommercially available Agaricus blazei fruit bodies were used instead ofTricholoma matsutake fruit bodies harvested in Iwate Prefecture, toobtain 3.6 g of dry powder of a hot water extract of Agaricus blazeifruit bodies.

Comparative Example 2

[0108] Preparation of Dry Powder of Hot Water Extract of Ganodermalucidum (Fr.) Karst Fruit Bodies

[0109] The procedures described in Example 4 were repeated, except thatcommercially available Ganoderma lucidum (Fr.) Karst fruit bodies wereused instead of Tricholoma matsutake fruit bodies harvested in IwatePrefecture, to obtain 2.4 g of dry powder of a hot water extract ofGanoderma lucidum (Fr.) Karst fruit bodies.

Comparative Example 3

[0110] Preparation of Dry Powder of Hot Water Extract of Lentinus edodes(Berk.) Sing. Fruit Bodies

[0111] The procedures described in Example 4 were repeated, except thatcommercially available Lentinus edodes (Berk.) Sing. fruit bodies wereused instead of Tricholoma matsutake fruit bodies harvested in IwatePrefecture, to obtain 1.4 g of dry powder of a hot water extract ofLentinus edodes (Berk.) Sing. fruit bodies.

Analytical Example 1

[0112] Identification of Tricholoma matsutake Strain FERM BP-7304 byRAPD Method

[0113] In the present analytical example, DNA patterns which wereamplified by a random amplified polymorphic DNA (RAPD) method using DNAof Tricholoma matsutake strain FERM BP-7304 according to the presentinvention and any one of six kinds of primers (10 mer) for a polymerasechain reaction (PCR) were compared with those of plural known Tricholomamatsutake strains.

[0114] As the known strains for comparison, 13 kinds of Tricholomamatsutake strains listed in Table 1 were used. Dried mycelia wereobtained, and then the resulting mycelia were crushed to obtain drypowder, in a similar manner as that described in Example 1. Yields ofmycelia of Tricholoma matsutake strains (weight of dried mycelia per 100mL of medium; an average of 10 samples) and origins (facilities in whichthe strains were established) of Tricholoma matsutake strains are shownin Table 1. TABLE 1 No. Strain Yield (g) Origin (Facility) 1 IFO 69150.67 Institute for Fermentation, Osaka 2 IFO 6925 0.50 Institute forFermentation, Osaka 3 IFO 6930 0.72 Institute for Fermentation, Osaka 4IFO 6935 0.65 Institute for Fermentation, Osaka 5 CM 627-2 0.79 KurehaChemical Industry Co. Ltd 6 CM 627-4 0.80 Kureha Chemical Industry Co.Ltd 7 IFO 30604 0.49 Institute for Fermentation, Osaka 8 IFO 30605 0.71Institute for Fermentation, Osaka 9 IFO 30606 0.35 Institute forFermentation, Osaka 10 MAFF 460039 0.56 National Institute ofAgrobiological Science; Ministry of Agriculture, Forestry and Fisheriesof Japan 11 KT 001 0.68 Kureha Chemical Industry Co. Ltd 12 IFO 69200.72 Institute for Fermentation, Osaka 13 IFO 6933 0.56 Institute forFermentation, Osaka

[0115] As the primers for PCR, the following primers: primer RAPD1, (SEQID NO: 1; TGGTCACCGA) primer RAPD2, (SEQ ID NO: 2; AGCGCCATTG) primerRAPD3, (SEQ ID NO: 3; TTCGAGCCAG) primer RAPD4, (SEQ ID NO: 4;TGCGTGCTTG) primer RAPD5, and (SEQ ID NO: 5; GACTAGCCTC) primer RAPD6(SEQ ID NO: 6; CTCACCGTCC)

[0116] were prepared by a chemical synthesis method.

[0117] The DNAs used in the RAPD method were prepared using acommercially available DNA preparation kit (Dneasy Plant Mini Kit;QIAGEN) by a method similar to that described in an attached manual[Dneasy Plant Mini Handbook for DNA isolation from plant tissue (March,1999, QIAGEN, K. K., Tokyo)] as described below.

[0118] After removing water roughly, each mycelia (approximately 100 mg)of Tricholoma matsutake strains was put into a tube, frozen with liquidnitrogen, and kept at −80° C. until using. Each tube containing myceliawas thawed, and then an AP1 buffer (400 μL) and an RNase A stocksolution (4 μL) were added to the tube. The whole was thoroughly mixedby a vortex mixer. The tube was incubated in a water bath at 65° C. for10 minutes. The tube was inverted several times to mix the contentsduring the incubation. After the incubation, an AP2 buffer (130 μL) wasadded to the mixture, and then the whole was allowed to stand on ice for5 minutes. The contents in the tube were applied to a column(QIAshredder spin column). The column was put into a 2 mL-test tube andcentrifuged at 15000 rpm for 2 minutes. After the centrifugation, asolution which had passed through the column and had gathered at thebottom of the tube (passage liquid) was carefully transferred to anothertest tube, so as not to touch cell pellets at the bottom of the tube. Avolume of the passage liquid was measured.

[0119] An equal volume of 100% ethanol and a half volume of a bufferAP3, with respect to the passage liquid, were added to the test tube andmixed thoroughly. After 650 μL of the mixture was applied to anothercolumn (DNeasy mini spin column), the column was put into a 2 mL-testtube and centrifuged at 8000 rpm for 1 minute. After the centrifugation,an AW buffer (500 μL) was applied to the column. The column was put intothe 2 mL-test tube and centrifuged at 8000 rpm for 1 minute, and then asolution collected at the bottom of the test tube was discarded. Afterthe AW buffer (500 μL) was applied to the column, the column was putinto the 2 mL-test tube and centrifuged at 15000 rpm for 2 minutes, andthen a solution collected at the bottom of the test tube was discarded.The washed column was put into a new test tube. After an AE buffer (100μL) kept at 65° C. was applied to the washed column, the column wasallowed to stand at room temperature for 5 minutes, and centrifuged at8000 rpm for 1 minute to collect a DNA solution at the bottom of thetest tube. The similar procedures were repeated once again to collectthe DNA solution (approximately 200 μL in total). A portion (20 μL) ofthe resulting DNA solution was electrophoresed on a 1% agarose gel todetermine an amount of DNA recovered and a purity of the DNA.

[0120] A PCR was carried out in a reaction solution (a total volume=25μL) shown in Table 2. In the PCR, a cycle consisting of treatments at94° C. for 30 seconds (a denaturing step), 36° C. for 1 minute (anannealing step), and 72° C. for 2 minutes (an elongation step) wasrepeated 45 times. In this connection, a preliminary denaturing step for2 minutes was performed before the first cycle, and a final elongationstep for 2 minutes was performed after the 45th cycle. The reactionsolution shown in Table 2 was prepared by mixing an Ex Taq™ buffer,TaKaRa Ex Taq™, and a Taq Start antibody, adding distilled water, a10×Ex Taq™ buffer, dNTP, and a primer to the mixture after 2 to 3seconds from the mixing treatment, and finally adding a DNA solution.TABLE 2 Reaction solution (per a tube) Ex Taq ™ buffer (Takara Shuzo)0.8 μL TaKaRa Ex Taq ™ (Takara Shuzo) 0.2 μL Taq Start antibody (TakaraShuzo) 0.2 μL Distilled water 8.3 μL 10 × Ex Taq ™ buffer (Takara Shuzo)2.5 μL dNTP 2.0 μL Primer 1.0 μL DNA  10 μL

[0121] DNA patterns obtained by separating the resulting PCR products byan agarose gel electrophoresis are shown in FIGS. 1 to 4. FIG. 1 showsDNA patterns of comparative strains of Nos. 1 to 6 in Table 1; FIG. 2shows DNA patterns of comparative strains of Nos. 7 to 11 in Table 1;FIG. 3 shows DNA patterns of comparative strains of Nos. 12 to 13 inTable 1 and Tricholoma matsutake strain FERM BP-7304 of the presentinvention; and FIG. 4 shows DNA patterns of Tricholoma matsutake strainFERM BP-7304 of the present invention.

[0122] Circled numbers “1” to “6” in FIG. 1, circled numbers “7” to “11”in FIG. 2, and circled numbers “12” to “13” in FIG. 3 correspond to thestrain Nos. 1 to 13 in Table 1, respectively. For example, lanesrepresented by the circled number “1” in FIG. 1 show results of thestrain No. 1 (IFO 6915) in Table 1. Further, lanes represented by thecircled number “14” in FIG. 3 show results of Tricholoma matsutakestrain FERM BP-7304 of the present invention.

[0123] Words “RAPD1” to “RAPD6” in FIGS. 1 to 3 mean primers RAPD1 toRAPD6, respectively.

[0124] Lanes 1 to 6 in FIG. 4 show results of Tricholoma matsutakestrain FERM BP-7304 of the present invention obtained using primersRAPD1 to RAPD6, respectively.

[0125] Further, a symbol “M” in FIGS. 1 to 4 means a DNA molecularweight marker. A single DNA molecular weight marker (1 kb DNA ladder,catalog No. 15415-018, Life Technologies, GIBCO-BRL, USA) was used inthe electrophoresis. In FIG. 4, a band represented by an arrow A shows aDNA fragment of 4072 bp; a band represented by an arrow B shows a DNAfragment of 3054 bp; a band represented by an arrow C shows a stack ofDNA fragments of 2036 bp and 1636 bp; a band represented by an arrow Dshows a DNA fragment of 1018 bp; and a band represented by an arrow Eshows a stack of DNA fragments of 517 bp, 506 bp, 396 bp, 344 bp, and298 bp.

[0126] As shown in FIGS. 1 to 4, DNA patterns of Tricholoma matsutakestrain FERM BP-7304 were different from those of 13 kinds of Tricholomamatsutake strains for comparison listed in Table 1.

[0127] Further, although DNA patterns are not actually shown, DNApatterns of Tricholoma matsutake strain FERM BP-7304 were different fromthose of 31 kinds of Tricholoma matsutake strains other than 13 kinds ofcomparative strains listed in Table 1. The 31 kinds of strains were MAFF460031, MAFF 460033, MAFF 460034, MAFF 460035, MAFF 460036, MAFF 460037,MAFF 460038, MAFF 460040, MAFF 460041, MAFF 460042, MAFF 460046, MAFF460050, and MAFF 460096 (National Institute of Agrobiological Science;Ministry of Agriculture, Forestry and Fisheries of Japan); CM 627-3, CM627-5, CM 627-6, and CM 627-7(Kureha Chemical Industry Co. Ltd); and IFO6929, IFO 6931, IFO 6932, IFO 6934, IFO 6916, IFO 6917, IFO 6918, IFO6919, IFO 6921, IFO 6922, IFO 6923, IFO 6924, IFO 6926, and IFO 6928(Institute for Fermentation, Osaka).

Analytical Example 2

[0128] GC Content of Tricholoma matsutake Strain FERM BP-7304

[0129] After 20 mL of a phosphate-buffered saline (PBS) solution (pH7.4)containing 1 mg/mL proteinase K (Merck, Germany) was added to a 50mL-conical flask containing mycelia (wet-base weight=1.0 g) ofTricholoma matsutake strain FERM BP-7304 prepared in a similar manner asthat described in Example 1, the whole was reacted at 65° C. for 2 hourswhile occasionally shaking. The reaction mixture was heated at 100° C.for 10 minutes to inactivate the enzyme, and cooled to 37° C. Anappropriate amount (approximately 1 mg) of Zymolyase (Seikagaku Corp.)was added to the reaction mixture, and the whole was reacted at 37° C.for 4 hours. After 40 mL of a Tris-SDS (sodium dodecyl sulfate) bufferwas added, mycelia was lysed at 60° C. to prepare a DNA extract.

[0130] An equal volume of phenol was added to the DNA extract. Aftermixing, the mixture was centrifuged to remove a phenol layer. Crude DNAwas recovered as pellets from the resulting supernatant by an ethanolprecipitation method, and washed with 70 to 90% ethanol. The resultingDNA pellets were dissolved in 5 mL of a citrate buffer, and then anRNase treatment was performed. A small amount of phenol was added to theRNase-treated solution. After mixing, the mixture was centrifuged toremove a phenol layer. Crude DNA was recovered from the resultingsupernatant by the ethanol precipitation method, and washed with 70 to90% ethanol. After the second RNase treatment was performed, 0.5 mL ofan EDTA(ethylenediaminetetraacetic acid)—containing acetate buffer wasadded. DNA was precipitated by adding propanol and recovered bycentrifugation. The resulting DNA was washed with 70 to 90% ethanol, andfinally dissolved in 1 mL of a citrate buffer to obtain a purified DNAsolution.

[0131] A portion (100 μL) of the resulting purified DNA solution washeated at 100° C. for 10 minutes, cooled on ice, and treated withnuclease P1 at 50° C. for 1 hour to obtain nucleotide products. A GCcontent was measured by a high performance liquid chromatograph (LC-6A;Shimadzu). A GC kit (Yamasa Corporation) was used as a standard, acolumn YMC-Pack AQ-312 (diameter=6.0 mm, length=150 mm) was used as acolumn, and a 0.2 mol/L ammonium phosphate solution (pH=approximately4.5) was used as a mobile phase. An amount of injection was 10 μL,detection was carried out at a wavelength of 270 nm, and a peak wasidentified by a retention time method. A modified percentage method wasused as a measuring method.

[0132] The GC content of Tricholoma matsutake strain FERM BP-7304 was49.9%.

Example for Evaluation 1

[0133] Evaluation for Activity of Promoting a Recovery from Stress

[0134] (1) Evaluation for Hot Water Extract and Alkaline SolutionExtract of Tricholoma matsutake Strain FERM BP-7304

[0135] In the present example for evaluation, a mixture of the drypowder of a hot water extract of Tricholoma matsutake strain FERMBP-7304 prepared in Example 2, and the dry powder of an alkalinesolution extract thereof prepared in Example 2 (a ratio of the drypowder of a hot water extract to that of alkaline solutionextract=3.2:5.1) was used as a sample for evaluation. Further, after thesample for evaluation was orally administered to mice for 4 weeks,restraint stress was loaded for 18 hours, and then a natural killer (NK)cell activity was measured after a release of the stress to examine theeffects of the sample.

[0136] More particularly, an aqueous solution of the sample forevaluation was orally administered to 8-week-old male C57BL/6 mice(purchased from Japan SLC; 5 to 10 mice per a group) at a dose of 50mg/kg/day for 4 weeks in normal breeding cages. The aqueous solution wasprepared by mixing the dry powder of a hot water extract of Tricholomamatsutake strain FERM BP-7304 and the dry powder of an alkaline solutionextract thereof, which were prepared in Example 2, at a ratio of3.2:5.1, and then dissolving the mixed dry powder in distilled water.

[0137] After the administration for 4 weeks, mice were transferred fromthe normal breeding cages to 50 mL-capped polypropylene centrifuge tubes(catalog No. 2341-050; Asahi Techno Glass Corporation) with air vents sothat a mouse was confined in a tube. The confined mice could not move inthe tubes. The tubes in which mice were confined were placed in thecages and allowed to stand for 18 hours to load the mice with restraintstress. After the stress loading for 18 hours, mice were transferredfrom the tubes to the breeding cages, and bred under ordinary breedingconditions.

[0138] After a predetermined number of days [0 day (immediately afterthe release), 1 day, 3 days, 5 days, 7 days, and 14 days] had passedfrom the release of the restraint stress, mice were sacrificed, and anatural killer (NK) cell activity was evaluated by measuring a cytotoxicactivity of lymphocytes against an NK-sensitive tumor cell strain YAC-1in vitro, in accordance with the following procedures.

[0139] Spleens and mesenterium lymph nodes were aseptically taken frommice and transferred to a sterile petri dish containing a Hanks balancedsalt solution. The lymph nodes were teased with scissors and tweezers,and passed through a mesh to prepare a suspension containing singlelymphocyte cells. The cells were washed three times with an RPMI 1640medium containing a 10% bovine fetal serum which had been heated at 56°C. for 30 minutes. Then, a concentration of cells was adjusted to5×10⁶/mL with an RPMI 1640 medium containing a 10% bovine fetal serumwhich had been heated at 56° C. for 30 minutes, 20 mmol/L of4-(2-hydroxyethyl)-1-piperazine ethanesulfonate, and 30 μg/mL ofgentamicin. The resulting cell suspension was used as an effector cell.

[0140] The YAC-1 cell used as a target cell was maintained in an RPMI1640 medium containing a 10% bovine fetal serum which had been heated at56° C. for 30 minutes, at Biomedical Research Laboratories, KurehaChemical Industry Co. Ltd. The YAC-1 cells were reacted with radioactivesodium chromate (Amersham Japan) at 37° C. for 20 minutes. Unreactedradioactive sodium chromate was removed by washing three times with anRPMI 1640 medium containing a 10% bovine fetal serum which had beenheated at 56° C. for 30 minutes, and a concentration of tumor cellslabeled with radioactive chromium was adjusted to 5×10⁴/mL.

[0141] 0.1 mL of the effector cell suspension or a double-diluted seriesthereof and 0.1 mL of the suspension of tumor cells labeled withradioactive chromium were put into a test tube, and reacted in a 5%carbon dioxide gas incubator at 37° C. 4 hours. In this connection, tocalculate a “specific lysis” described below, a suspension prepared byputting the tumor cells labeled with radioactive chromium and a mediuminto a test tube, and a suspension prepared by putting the tumor cellslabeled with radioactive chromium and a detergent (Triton; a finalconcentration=0.05%) into a test tube were also reacted in a 5% carbondioxide gas incubator at 37° C. for 4 hours. After the reaction wascompleted, 1.5 mL of an RPMI 1640 medium containing a 10% bovine fetalserum, which had been heated at 56° C. for 30 minutes, was added to eachtest tube, and thoroughly mixed by a mixer. The whole was centrifuged at12,000 rpm for 5 minutes at 4° C. to obtain a supernatant, and aradioactivity was measured by a gamma counter.

[0142] A specific lysis (S.L.) was calculated by an equation:

[S.L.]={(B-B _(f))/(B _(max) −B _(f))}×100

[0143] wherein S.L. is a specific lysis (unit=%), B is a radioactivity(unit=Bq) of a supernatant of an experimental group, B_(f) is aradioactivity (unit=Bq) of a supernatant of a spontaneously releasinggroup, and B_(max) is a radioactivity (unit=Bq) of a supernatant of amaximum releasing group. The spontaneously releasing group means a groupof culturing only the tumor cells labeled with radioactive chromium, andthe maximum releasing group means a group of culturing tumor cellslabeled with radioactive chromium treated with Triton. The NK cellactivity was represented by “Lytic Units 30% (LU30)”, that is, a numberof cells which kill 30% tumor cells per 10⁷ cells of effector cells.

[0144] The results are shown in FIG. 5. As a control (a normal group),the above procedures were repeated, except that distilled water wasorally administered for 4 weeks, instead of the aqueous solution of thesample for evaluation, and that the restraint stress for 18 hours wasnot loaded. Further, as a test for comparison, the above procedures wererepeated, except that distilled water was orally administered for 4weeks, instead of the aqueous solution of the sample for evaluation.

[0145] In FIG. 5, line a (white square) shows a result of the control (anormal group; without stress), line b (black circle) shows a result whenthe aqueous solution of the sample for evaluation (the mixture of thedry powder of a hot water extract of Tricholoma matsutake strain FERMBP-7304 and the dry powder of an alkaline solution extract thereof, at aratio of 3.2:5.1) according to the present invention was administered,and line c (black triangle) shows a result of the test for comparison(administration of distilled water and stress loading). Further, in FIG.5, the symbol “+” above black circles in line b means P<0.01 (withrespect to a result represented by black triangles in line c), and thesymbol “*” above black triangles in line c means P<0.01 (with respect toa result represented by white squares in line a).

[0146] As shown in lines b and c in FIG. 5, the NK cell activity isremarkably lowered by loading restraint stress. As shown in line c,after the release of the restraint stress, the NK cell activity wasslowly recovered without any treatment. As shown in line c, the recoveryof the NK cell activity was significantly promoted by previouslyadministering the mixture of the dry powder of a hot water extract ofTricholoma matsutake strain FERM BP-7304 and the dry powder of analkaline solution extract thereof, at a ratio of 3.2:5.1.

[0147] (2) Evaluation for Dry Powder of Tricholoma matsutake Strain FERMBP-7304, Dry Powder of Tricholoma matsutake Fruit Bodies from IwatePrefecture, and Mixture of Dry Powder of a Hot Water Extract ofTricholoma matsutake Fruit Bodies from Iwate Prefecture and Dry Powderof an Alkaline Solution Extract thereof

[0148] The procedures in Example for evaluation 1(1) were repeatedexcept that the dry powder (prepared in Example 1; 50 mg/kg/day) ofTricholoma matsutake strain FERM BP-7304, the dry powder (prepared inExample 3; 50 mg/kg/day) of commercially available Tricholoma matsutakefruit bodies harvested in Iwate Prefecture, and the mixture (25mg/kg/day) of dry powder (prepared in Example 4) of hot water extract ofcommercially available Tricholoma matsutake fruit bodies harvested inIwate Prefecture and dry powder (prepared in Example 4) of alkalinesolution extract thereof at a ratio of 1.0:5.1 were used, instead of themixture of the dry powder of a hot water extract of Tricholoma matsutakestrain FERM BP-7304 and the dry powder of an alkaline solution extractthereof at a ratio of 3.2:5.1.

[0149] The results are shown in Table 3. The symbol “*” in Table 3 meansp<0.01 (with respect to administration of distilled water). TABLE 3 NKcell activity (LU30) 0 day 1 day 3 days 5 days 7 days 14 days Control 4947 48 50 48 48 (without stress loading) Test for comparison 7  9 11 2230 42 (administration of distilled water and stress loading) Example 110  21*  35*  46*  48* 50 Example 3 10  20*  31*  37*  49*  51* Example4 11  22*  37*  41*  46* 48

[0150] (3) Evaluation for Dry Powder of Tricholoma matsutake Strains

[0151] The procedures in Example for evaluation 1(1) were repeatedexcept that the dry powder (prepared in Example 1; 50 mg/kg/day) ofTricholoma matsutake strain FERM BP-7304, and each dry powder (50mg/kg/day) of 13 kinds of Tricholoma matsutake strains listed in Table 1and used in Analytical Example 1 were used, instead of the mixture ofthe dry powder of a hot water extract of Tricholoma matsutake strainFERM BP-7304 and the dry powder of an alkaline solution extract thereofat a ratio of 3.2:5.1.

[0152] The results are shown in Table 4 and FIGS. 6 to 9.

[0153] The symbol “*” in Table 4 means p<0.01 (with respect toadministration of distilled water).

[0154] In FIGS. 6 to 9, line a (black square) shows a result of thecontrol (without stress), and line b (white square) shows a result ofthe test for comparison (administration of distilled water and stressloading).

[0155] In FIG. 6, line c (black triangle) shows a result of Tricholomamatsutake strain FERM BP-7304 of the present invention, line d (blacklozenge) shows a result of Tricholoma matsutake strain IFO 6915, andline e (black circle) shows a result of Tricholoma matsutake strain IFO6925.

[0156] In FIG. 7, line f (black triangle) shows a result of Tricholomamatsutake strain IFO 6930, line g (black lozenge) shows a result ofTricholoma matsutake strain IFO 6935, line h (black circle) shows aresult of Tricholoma matsutake strain CM 627-2, and line i (whitecircle) shows a result of Tricholoma matsutake strain CM 627-4.

[0157] In FIG. 8, line j (black triangle) shows a result of Tricholomamatsutake strain IFO 30604, line k (black lozenge) shows a result ofTricholoma matsutake strain IFO 30605, line l (black circle) shows aresult of Tricholoma matsutake strain IFO 30606, and line m (whitecircle) shows a result of Tricholoma matsutake strain MAFF 460039.

[0158] In FIG. 9, line n (black triangle) shows a result of Tricholomamatsutake strain KT 001, line p (black lozenge) shows a result ofTricholoma matsutake strain IFO 6920, and line q (black circle) shows aresult of Tricholoma matsutake strain IFO 6933. TABLE 4 NK cell activity(LU30) Strains 0 day 1 day 3 days 5 days 7 days 14 days Control 48 47 4646 47 46 (without stress loading) Test for comparison 6  8 14 21 28 43(administration of distilled water and stress loading) [Examples] FERMBP-7304 11  25*  42*  47*  50* 48 IFO 6915 7 15 18 31 38 45 IFO 6925 917 19 37 41 43 IFO 6930 8 10 18 29 36 45 IFO 6935 8 13 21 32 39 42 CM627-2 7 14 20 33 44 44 CM 627-4 8 13 19 35 41 46 IFO 30604 6 11 20 35 3940 IFO 30605 6 14 18 36 38 45 IFO 30606 6 14 18 30 36 44 MAFF 460039 813 19 29 37 45 KT 001 7 15 21 34 42 41 IFO 6920 7 11 19 33 40 40 IFO6933 7 10 17 30 37 42

[0159] As shown in Table 4, the dry powder of Tricholoma matsutakestrain FERM BP-7304, and each dry powder of 13 kinds of Tricholomamatsutake strains listed in Table 1 promoted the recovery of the NK cellactivity. Particularly, the dry powder of Tricholoma matsutake strainFERM BP-7304 exhibited the most excellent activity of promoting arecovery from stress.

[0160] Further, The procedures in Example for evaluation 1(1) wererepeated except that each dry powder (50 mg/kg/day) of 31 kinds ofTricholoma matsutake strains other than the 13 (39) kinds of Tricholomamatsutake strains listed in Table 1 were used. The 31 kinds of strainswere MAFF 460031, MAFF 460033, MAFF 460034, MAFF 460035, MAFF 460036,MAFF 460037, MAFF 460038, MAFF 460040, MAFF 460041, MAFF 460042, MAFF460046, MAFF 460050, and MAFF 460096 (National Institute ofAgrobiological Science; Ministry of Agriculture, Forestry and Fisheriesof Japan);

[0161] CM 627-3, CM 627-5, CM 627-6, and CM 627-7(Kureha ChemicalIndustry Co. Ltd); and

[0162] IFO 6929, IFO 6931, IFO 6932, IFO 6934, IFO 6916, IFO 6917, IFO6918, IFO 6919, IFO 6921, IFO 6922, IFO 6923, IFO 6924, IFO 6926, andIFO 6928 (Institute for Fermentation, Osaka). As a result, the recoveryof the NK cell activity was confirmed in these Tricholoma matsutakestrains, but no strains exhibited a more excellent activity of promotinga recovery from stress than that of Tricholoma matsutake strain FERMBP-7304.

[0163] (4) Evaluation for Dry Powder of Hot Water Extract of Tricholomamatsutake Fruit Bodies from Nagano Prefecture and Dry Powder of HotWater Extract of Various Mushrooms

[0164] The procedures in Example for evaluation 1(1) were repeatedexcept that the dry powder (prepared in Example 5) of hot water extractof Tricholoma matsutake fruit bodies harvested in Nagano Prefecture, oreach dry powder (prepared in Comparative Examples 1 to 3, respectively)of hot water extract of Agaricus blazei fruit bodies, Ganoderma lucidum(Fr.) Karst fruit bodies, or Lentinus edodes (Berk.) Sing. fruit bodieswere used as samples for evaluation. The dose was 250 mg/kg/day in allcases.

[0165] The results are shown in Table 5. In Table 5, the symbol “*”means p<0.01 (with respect to administration of distilled water). TABLE5 NK cell activity (LU30) 0 day 1 day 3 days 5 days 7 days 14 daysControl 47 48 48 49 46 47 (without stress loading) Test for comparison 6 8 13 26 29 44 (administration of distilled water and stress loading)Example 5 10  14*  22*  34*  37* 47 Comparative Examples 1 5  7 15 27 3037 2 3  8 11 24 28 36 3 4  9 10 27 29 40

[0166] Industrial Applicability

[0167] According to the pharmaceutical composition of the presentinvention for promoting a recovery from stress, the recovery from stresscan be promoted.

[0168] Free Text in Sequence Listing

[0169] Features of “Artificial Sequence” are described in the numericidentifier <223> in the Sequence Listing. More particularly,oligonucleotides consisting of the base sequences of SEQ ID NOS: 1 to 6are a primer RAPD1 to a primer RAPD6, respectively.

[0170] As above, the present invention is explained with reference toparticular embodiments, but modifications and improvements obvious tothose skilled in the art are included in the scope of the presentinvention.

1 6 1 10 DNA Artificial Sequence Description of Artificial Sequenceprimer RAPD1 1 tggtcaccga 10 2 10 DNA Artificial Sequence Description ofArtificial Sequence primer RAPD2 2 agcgccattg 10 3 10 DNA ArtificialSequence Description of Artificial Sequence primer RAPD3 3 ttcgagccag 104 10 DNA Artificial Sequence Description of Artificial Sequence primerRAPD4 4 tgcgtgcttg 10 5 10 DNA Artificial Sequence Description ofArtificial Sequence primer RAPD5 5 gactagcctc 10 6 10 DNA ArtificialSequence Description of Artificial Sequence primer RAPD6 6 ctcaccgtcc 10

1. A pharmaceutical composition for promoting a recovery from stress,comprising Tricholoma matsutake, a hot water extract of Tricholomamatsutake or a dried product thereof, or an alkaline solution extract ofTricholoma matsutake or a dried product thereof, and a pharmaceuticallyacceptable carrier.
 2. The pharmaceutical composition for promoting arecovery from stress according to claim 1, wherein the Tricholomamatsutake is a Tricholoma matsutake strain FERM BP-7304.
 3. Thepharmaceutical composition for promoting a recovery from stressaccording to claim 1 or 2, wherein the Tricholoma matsutake is amycelium, a broth, or a fruit body.
 4. A functional food for promoting arecovery from stress, comprising Tricholoma matsutake, a hot waterextract of Tricholoma matsutake or a dried product thereof, or analkaline solution extract of Tricholoma matsutake or a dried productthereof, alone or optionally with one or more food components.
 5. Thefunctional food for promoting a recovery from stress according to claim4, wherein the Tricholoma matsutake is a Tricholoma matsutake strainFERM BP-7304.
 6. The functional food for promoting a recovery fromstress according to claim 4 or 5, wherein the Tricholoma matsutake is amycelium, a broth, or a fruit body.
 7. An oral hygienic composition forpromoting a recovery from stress, comprising Tricholoma matsutake, a hotwater extract of Tricholoma matsutake or a dried product thereof, or analkaline solution extract of Tricholoma matsutake or a dried productthereof, and a carrier for an oral hygienic composition.
 8. The oralhygienic composition for promoting a recovery from stress according toclaim 7, wherein the Tricholoma matsutake is a Tricholoma matsutakestrain FERM BP-7304.
 9. The oral hygienic composition for promoting arecovery from stress according to claim 7 or 8, wherein the Tricholomamatsutake is a mycelium, a broth, or a fruit body.
 10. A method forpromoting a recovery from stress, comprising administering to a subjectin need thereof Tricholoma matsutake, a hot water extract of Tricholomamatsutake or a dried product thereof, or an alkaline solution extract ofTricholoma matsutake or a dried product thereof, in an amount effectivetherefor.
 11. The method for promoting a recovery from stress accordingto claim 10, wherein the Tricholoma matsutake is a Tricholoma matsutakestrain FERM BP-7304.
 12. The method for promoting a recovery from stressaccording to claim 10 or 11, wherein the Tricholoma matsutake is amycelium, a broth, or a fruit body.
 13. Use of Tricholoma matsutake, ahot water extract of Tricholoma matsutake or a dried product thereof, oran alkaline solution extract of Tricholoma matsutake or a dried productthereof, in the manufacture of a pharmaceutical composition forpromoting a recovery from stress, a functional food for promoting arecovery from stress, or an oral hygienic composition for promoting arecovery from stress.
 14. The use according to claim 13, wherein theTricholoma matsutake is a Tricholoma matsutake strain FERM BP-7304. 15.The use according to claim 13 or 14, wherein the Tricholoma matsutake isa mycelium, a broth, or a fruit body.
 16. A Tricholoma matsutake strainFERM BP-7304.
 17. A mycelium, a broth, or a fruit body of a Tricholomamatsutake strain FERM BP-7304.
 18. A hot water extract of a Tricholomamatsutake strain FERM BP-7304 or a dried product thereof, or an alkalinesolution extract of a Tricholoma matsutake strain FERM BP-7304 or adried product thereof.
 19. The hot water extract or the dried productthereof, or the alkaline solution extract or the dried product thereofaccording to claim 18, wherein the Tricholoma matsutake strain FERMBP-7304 is a mycelium, a broth, or a fruit body.